CHE.496/2009/Responses/a9

Genetic circuit engineering (part 1)

• Discussion leader: Thaddeus

Rohini's Response

A Synthetic Oscillator Network of Transcriptional Regulators

• Repressilator (“artificial clock”): a system consisting of three transcriptional repressors that create an oscillating network
• The network periodically induces the synthesis of GFP depending on its state in individual cells
• In the experiment, the scientists implemented the repressilator into E. coli. They observed that the oscillations were slower than the cell division cycle and displayed noisy behavior.
• How the repressilator works:the first repressilator protein (LacI) inhibits the transcription of the second repressor gene (tetR) whose protein product inhibits the expression of the third gene (cI).
• The repressilator runs on a negative feedback loop.
• The network depends on: the transcription rate on repressor concentration, translation rate and the decay rates of the protein and messenger RNA.
• Oscillations are favored when there are strong promoters coupled to efficient ribosome-binding sites, tight transcriptional repression, cooperative repression characteristics and comparable protein and mRNA decay rates.
• To make the artificial network exhibit oscillatory behavior: use strong and tightly repressible hybrid promoters and match the lifetime of the effective repressor protein closer to that of mRNA.
• Experiment:

-studied the repressilator by isolating single cells under the microscope and monitoring their florescence intensity

-results: the state of the network is transmitted to the progeny cells, stochastic effects produce noise

-next step: varying the host species and genetic background that the repressilator is inserted into

Construction of a genetic toggle switch in E. coli

• Examples of specialized gene circuits: Cyanobacteria circadian oscillator, bacteriophage lamda switch
• Genetic toggle switch-a synthetic, bistable gene-regulatory network in E.coli constructed from two repressible promoters arranged in a mutually inhibitory network and two promoters. Each promoter is inhibited by the repressor that is transcribed by the opposing promoter. By changing the chemical or thermal induction the toggle can be flipped between stable states.

I really enjoyed the reading assignment as it provided me with a better understanding of how synthetic oscillatory networks work. For my literature review assignment, I am reading about the VGEM group in Taiwan who came up with a capsule designed to filter blood and replace the function of the kidney. The capsule will be used by patients that are currently using hemodialysis machines. The capsule is ingested and then passes through the digestive track until it comes to the small intestine. In the small intestine, it has an attachment anchor latching on to the epithelial cells. The capsule contains a clearance device that filters out urea, phosphate and other substances from the blood. The capsule remains attached to the small intestine for a fixed amount of time controlled by time regulating oscillators. The team experimented with two systems, cyanoxilator and reloxilator. The relaxilator is based on the lysogenic genetic switch in phage lambda and the cyanoxilator is constructed from three proteins found in cyanobacteria. When time has expired, the capsule detaches and exits the body. The project showed how useful these oscillatory networks can be in creating innovative treatments for medical conditions.

Rohini Manaktala 17:24, 15 March 2009 (EST)

Thaddeus's Response

Repressilator paper

This paper describes the design principles used to create the repressilator.

• Repressilator is composed of three repressor promoter pairs.
• The repressor cycle lasts longer than the cell cycle so state must be inheritable.

System is influenced by

• dependence of transcriptional rate on repressor concentration
• Translation rate.
• Decay rates of protein and mRNA.

To optimize system they wanted

• Strong promotion/repression
• Cooperative repression.
• Comparable protein mRNA decay rates.

• They added a tag to decrease protein lifespan so that the two repressors had similar lifespans.
• Oscillations were tracked by isolating a single cell under a microscope and watching the progeny.
• The system was heavily influenced by stochastic effects.
• This paper was useful because it introduced the design considerations to keep in mind when making the genetic circuit.

Construction of toggle switch in E. Coli

• To present construction of a bistable toggle switch and lay out the simple supporting theory.
• Two constitutive promoters promote for cross repressing repressors.
• Any repressors whose activity can be blocked by a n inducer may be used.
• Act in trans
• Reporter GFP is cistronic to one circuit element.
• Presence of inducers could flip state of toggle switch.
• Elimination of repressor proteins from cell limits switching time.

Thaddeus Webb 19:51, 15 March 2009 (EDT)

Maria

“a synthetic oscillatory network of transcriptional regulators”

• repressilator=design of oscillating network from 3 novel transcriptional repressor systems
• cycle(150min) slower than cell division(~50min)- must pass to next generation
• noisy
• dependence of transcriptional rate of repressor concentration

** transcriptional rate

• • decay rate of protein
• can converge to steady state
• or be unstable- oscillate forever
• unknown parameters is a major design hurdle
• can use tags for proteases to diminish half-life of GFP
• stochasticity may account for noise

Toggle Switch

• mutually inhibitory network of 2 repressible promotors
• flipped by transient chemical or thermal induction
• doesn’t flip randomly
• can be delivered in 2 plasmids

• Maria Fini 01:09, 16 March 2009 (EDT):

Patrick's Response

• A synthetic oscillatory network of transcriptional regulators
  • The purpose of this article was to describe the design and construction of a genetic network in E. Coli that functions like an oscillatory circuit that is called the repressilator. This was achieved by coupling 3 repressor genes, where one repressor gene would repress a second repressor gene, and the third repressor gene would repress the first repressor in a negative cyclic feed-back loop as illustrated in Fig 1a. One could easily develop a biofilm to flash many different colors uniformly if a multi-repressor network was tied to the quorum-sensing network of bacteria. The different fluorescent proteins would be tied to the expression of a single repressor as shown in the figure for GFP that is tied to TetR. The behavior of the system is well-defined despite noise from stoichastic effects, the rate equations for protein concentrations and mRNA concentrations for a total of six differential equations that model the behavior of the system as shown in box 1. Furthermore, the equations were parameterized to include stoichastic effects based on experimental data to build a better model of the system. This is interesting to me since I am taking a class on reactions and I can relate to this subject matter far more readily than the previous semester.
• Construction of a genetic toggle switch in E. coli
  • The purpose of the article was to describe the construction of a genetic network that functions as a toggle switch that is controlled via 2 repressors, 2 promoters, and 2 inducers with one reporter gene as shown in figure 1. As reported by the researchers, the toggle switch is robust (has bistable behavior over a wide range of parameters and is tolerant of fluctuations in gene expression). In other words, this is a perpetual on-off switch that will express a fluorescent protein and then not express the protein. The selection of repressors and inducers that are able to alternate in a constant alternating sequence is governed by the rate equations in box 1. Interestingly, this system was modeled before it was constructed in order to determine which inducer/repressor best suited the model. This leads credence to the idea that theoretical design of complex and practical networks is possible.
• Patrick Gildea 18:22, 16 March 2009 (EDT):